Protein kinase C (PKC) has a prominent role in signal transduction of many bioactive substances. We synthesized the fluorescent derivative, phorbol-13-acetate-I 2-N-methyl-N-4-(N,N'-di(2-hydroxyethyl)amino)-7- nitrobenz-2-oxa-I ,3-diazole-aminododecanoate (N-C,,-Ac( 1 3)) of 12-0-tetradecanoylphorbol-I 3-acetate (TPA) to monitor the location of phorbol ester binding sites and evaluate its potential use as a probe of PKC in viable cells. The excitation maximum wavelength of N-C12-Ac(1 3) is close to 488 nm, facilitating its use in argon-ion laser flow and imaging cytometry. When incubated with 100 nM N-C,,-Ac(1 3) at 25"C, P,HR-l Burkitt lymphoma cells accumulated the dye rapidly, reaching maximum fluorescence within 25 min, 20-fold above autofluorescence. Addition of unlabeled TPA significantly decreased the fluorescence of N-C,,-Ac(l3) stained cells in a dose-dependent manner indicating specific displacement of the bound fluoroprobe. Competitive displacement of [3H]-phorbol-l 2,13-dibutyrate ([,HI-PBu,) from rat brain cytosol with N-C12-Ac(1 3) gave an apparent dissociation constant (K,) of 11 nM. N-Cl,-Ac(13) possessed biological activity similar to TPA. Like TPA (final concentration 65 nM) N-Cl,-Ac(l3), at a lower concentration (51 nM), induced expression of Epstein-Barr viral glycoprotein in P,HR-1 cells, differentiation of promyelocytic HL60 cells, and caused predicted changes in the mitotic cycle of histiocytic DD cells. Microspectrofluorometric images of single cells labeled with N-C1,-Ac(l3) showed bright fluorescence localized intracellularly and dim fluorescence in the nuclear region, consistent with dye binding mainly to cytoplasmic structures and/or organelles and being mostly excluded from the nucleus. Because of the high level of non-specific binding of N-C,,-Ac(l3), this probe i s not ideal for visualizing PKC in intact cells, but would be a valuable fluoroprobe to investigate the kinetic properties of purified PKC. Also, knowledge gained from these studies allows us to predict structures of fluorescent phorbols likely to have less non-specific binding and, consequently, be potentially useful for monitoring PKC in viable cells.